Luminescence Detection Of The Novel HOCl Fluorescent Probe In Cells And Preliminary Study Of Its Effect On A549 Cells Growth

Background and ObjectiveReactive oxygen species(ROS)is a byproduct in aerobic respiration metabolism,including superoxide anion,hydrogen peroxide and hydroxyl radicals,which have different targets in the body organism.ROS is not only able to induce the development of disease with the damaging of lipids,proteins and DNA,but also act as a signal molecule to regulate the physiological and pathological processes.Hypochlorous acid(HOCl)is one of the important ROS in human body,which mainly generated by myeloperoxidase and vascular peroxidase-land react chemically with a variety of substances such as protein,lipid nucleic acid and so on.HOCl plays an important role in human immune function system and the concentration will be gone up in the inflammation area,and promoting to destroy the invasive bacteria and pathogens.However,excessive HOCl or hypochlorite will induce tissue damage and the development of a range of diseases such as arteriosclerosis,arthritis and even cancer.Therefore,it is necessary to design and synthesize a novel and highly sensitive HOCl fluorescent probe to establish a fluorescence microscopy method for intracellular HOCl,so as to achieve real-time monitoring of changes on concentration of HOCl in cells.However,the ratio fluorescence probe which used to detect HOCl is still very few.Most of the reported HOCl fluorescent probes only contained single emission spectra,and the fluorescence signals are easily disturbed by environmental conditions such as probe concentration and instrument sensitivity.Wherefore,it is of great scientific significance and application prospect to design and synthesize a series of novel HOCl fluorescent probes.With the increase of morbidity and mortality,cancer has become a major cause of death and major public health problems in our country.During 2015,more than 4 million new cases of cancer and 3 million cancer deaths occured in China,among them lung cancer was the most common cause of cancer and the leading cause of cancer death.The latest global cancer statistics also showed that there were about 1.41 million new cancer cases and 8.2 million patients of them died of cancer in the world during 2012.Of which about 57%of cancer patients and 65%of cancer deaths are from developing countries.Whether developed or developing,lung cancer is still the first cause of cancer in men.Pneumonia is a key feature in the early stages of lung cancer and many lower respiratory tract diseases.It is mainly due to the large number of allergens,microbial immobilization,environmental pollutants inhaled into the lungs and smoking.At the same time pneumonia will cause phagocytic cell aggregation,resulting in a large number of bactericidal substances.The ROS and active nitrogen(RNS)of products play a key role.In addition,there are MPO in the lung inflammatory site.Abnormal concentrations of HOCl can induce apoptosis or cause cell necrosis directly.The study found that HOCl or MPO/H2O2/Cl-system can increase the levels of 3-chloro tyrosine and lipid peroxidation products in lung cells or in vivo lung tissue.HOCl can induce mutations in A549 cells.Therefore,to inhibit or treat lung cancer that called "the first cancer of cancer",we urgently need to develop some new drugs to resistant lung cancer cell proliferation.In this paper,a series of novel probes were synthesized for detection of HOCl.And we preliminarily studied the mechanism that A549 cells growth was inhibited by the probe in the method of chemical genetics.It can provide a new idea for the development of anti-cancer drugs,and supply new clues to hand for the new drug targets.Research methods1)The luminescence effect and light stability of HOCl probe in cells were detected by scanning confocal microscopy;2)We observed the cell morphology using the inverted phase contrast microscope;3)Cell viability was detected by the SRB;4)LDH method to detect whether cell death is necrosis;5)We verified whether cell death is apoptosis by Hoechst 33258 staining;6)The nuclear fragmentation was observed by AO staining;7)The cleaved PARP levels was analyzed in Western blotting;8)TUNEL staining was performed to detect apoptosis;9)The effect of CAN on tumor formation and angiogenesis in Chick chorioallantoic membrane(CAM)was analyzed by stereomicroscope;10)The Nrf2 activity was detected by the firefly luciferase reporter gene assay kit;11)We detected the level of HOCl by myeloperoxidase(MPO)Activity Assay Kit.Results1)CAN and other novel HOCl fluorescent probe in the cells showed a good photogenetic effect and light stability;2)The probe was able to detect the level of HOCl in RAW264.7 cells;3)The probe CAN is not co-located with mitochondria and lysosomes;4)There is no effect on the proliferation of human umbilical vein endothelial cells treated with CAN;5)CAN could induce A549 cells apoptosis in vitro;6)CAN inhibited tumor growth by inducing apoptosis in CAM;7)Nrf2 enzyme activity was improved by CAN in A549 cells.Conclusions Using RAW264.7 cells,we have demonstrated that a series of novel HOCl fluorescent probes in CAN have good luminescence and light stability in cells.Probes,in addition to CAN,could be used to detect endogenous HOCl levels in RAW264.7 cells.But CAN could significantly inhibit the growth of A549 cells and induce apoptosis,but there is no effect on the survival rate of vascular endothelial cells.Further,using chicken embryo allantoic membrane as the model,we had detemined that CAN by TUNEL staining could control the growth of tumor cells by apoptosis in vivo.But there was no effect no normal angiogenesis in CAM.In the end,we examined the effect of CAN on Nrf2 activity and the level of HOCl.The results showed that CAN down-regulated HOCl level by targeting HOCl or influencing MPO activity.In addition,CAN increased the activity of Nrf2,which indicated that CAN could up-regulat Nrf2 activity to aggravate ROS imbalances or influence the modification of HOCl though the increasing level of HOCl,so as to induce cell apoptosis finally.